THE EFFECT OF OESTRADIOL-17β ON ENZYMES CONCERNED WITH METABOLISM OF CARBOHYDRATE IN HUMAN ENDOMETRIUM IN VITRO

1969 ◽  
Vol 44 (1) ◽  
pp. 63-NP ◽  
Author(s):  
E. W. WILSON
Keyword(s):  
Enzyme Activity ◽  
Actinomycin D ◽  
Human Endometrium ◽  
Enzyme Protein ◽  
Quantitative Histochemistry ◽  
Qualitative And Quantitative ◽  
Hormonal Effect ◽  
Lactate Dehydrogenases ◽  
Regenerative Phase

SUMMARY Human endometrium in the regenerative phase was maintained for 5 hr. in vitro with oestradiol-17β alone or together with actinomycin D. Qualitative and quantitative histochemistry of the tissue showed that the activities of glucose-6-phosphate, 6-phosphogluconate, and lactate dehydrogenases were increased by oestradiol, and that actinomycin suppressed the hormonal effect. The activities of succinate and iso-citrate dehydrogenases were unaffected by oestradiol. An attempt is made to correlate the metabolic roles of the enzymes affected by oestradiol. The suppression, by actinomycin, of the oestradiol effect suggests that the increased enzyme activity is due to the formation of new enzyme protein.

scholarly journals Diamine-induced inhibition of liver ornithine decarboxylase

1979 ◽  
Vol 177 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Arja Kallio ◽  
Monica Löfman ◽  
Hannu Pösö ◽  
Juhani Jänne
Keyword(s):  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Decarboxylase Activity ◽  
Enzyme Protein ◽  
Intracellular Degradation ◽  
Ornithine Decarboxylase Activity ◽  
Normal Rat

Re!peated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ‘antienzymes’ [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858–1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme–inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

scholarly journals THE EFFECT OF CHANGES IN THE RESPIRATORY METABOLISM UPON GENOME ACTIVITY

1972 ◽  
Vol 55 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Hans J. Leenders ◽  
Pieter J. A. Beckers
Keyword(s):  
Enzyme Activity ◽  
Salivary Glands ◽  
Actinomycin D ◽  
Maximum Size ◽  
Respiratory Metabolism ◽  
Maximum Increase ◽  
Drosophila Hydei ◽  
Nicotinamide Adenine Dinucleotide Dehydrogenase ◽  
Genome Activity

The in vitro regression of experimentally induced chromosome puffs was investigated in explanted salivary gland chromosomes of Drosophila hydei. It was observed that the regression of the puffs 2-32A, 2-36A, 2-48C, and 4-81B is accelerated if substrates for the respiratory metabolism are supplied to the cells. A similar effect can be produced by addition of KCN or oligomycin to medium in which intact salivary glands are incubated. The acceleration of puff regression by these substances occurs not only if the puff-inducing stimulus is removed but as well under conditions in which the stimulus is maintained. Regression of the puffs 2-32A, 2-36A, and 4-81B is inhibited if cycloheximide is present in the incubation medium. Chloramphenicol has no effect on puff regression. Measurements on nicotinamide adenine dinucleotide-dehydrogenase activity in homogenates of salivary glands revealed an increase in enzyme activity of 41 %. Maximum increase is attained at 30 min after the induced puffs have reached their maximum size. The increase in enzyme activity does not occur if the glands are kept in a medium containing either actinomycin D or cycloheximide. Chloramphenicol does not inhibit the increase in enzyme activity. The possible relationship between puff activity and its control as a result of changes in the respiratory metabolism is discussed.

THE DEVELOPMENT OF AN IN VITRO SYSTEM FOR ESTIMATING OXYTOCINASE IN THE RABBIT HYPOTHALAMUS AND THE EFFECTS OF SOME PROTEIN SYNTHESIS INHIBITORS AND OESTRADIOL-17β ON ENZYME ACTIVITY

1974 ◽  
Vol 75 (3) ◽  
pp. 443-451 ◽  
Author(s):  
Dona A. Frith ◽  
K. C. Hooper
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Steroid Hormones ◽  
Actinomycin D ◽  
Protein Synthesis Inhibitors ◽  
In Vitro System

ABSTRACT An in vitro system for investigating the effects of steroid hormones and protein synthesis inhibitors on hypothalamic peptidases inactivating oxytocin has been developed. In the presence of oestradiol-17β enzyme activity was increased in the in vitro system whilst this increase was blocked completely by cycloheximide and partially blocked by actinomycin-D. It is apparent therefore that oestradiol-17β acts directly on the hypothalamus stimulating oxytocinase activity.

scholarly journals TEMPORAL ORDER IN MAMMALIAN CELLS

1969 ◽  
Vol 43 (2) ◽  
pp. 207-219 ◽  
Author(s):  
Robert R. Klevecz
Keyword(s):  
Enzyme Activity ◽  
Mammalian Cells ◽  
Temporal Order ◽  
Actinomycin D ◽  
Chinese Hamster ◽  
Enzyme Synthesis ◽  
S Phase ◽  
Selection System ◽  
Enzyme Protein ◽  
Chinese Hamster Cells

Chinese hamster cells were synchronized by the Colcemid-selection system. In cells with a division cycle time of 11.5–12 hr, the activity of the enzyme lactate dehydrogenase (LDH) underwent marked oscillations with a 3.5-hr period. Precipitation of labeled LDH enzyme with specific antibody indicated that the enzyme activity changes were the result of intermittent enzyme synthesis and relatively constant degradation. Inhibition of normal DNA replication with 4 mM of thymidine, while reducing the amount of new enzyme synthesized, did not prevent oscillations from occurring. Similarly, actinomycin D (AcD) added at the time of synchronization allowed some new enzyme synthesis to proceed in an oscillatory manner. LDH synthesis went on at nearly normal rates when AcD was added in the middle of S phase. However, addition of cycloheximide to cultures at any time in the cycle caused an immediate drop in levels of activity and in enzyme protein. The half-life of LDH, calculated either from loss of enzyme activity or precipitable radioactivity in cycloheximide-treated cultures, was between 2 and 2.5 hr.

scholarly journals Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver

1983 ◽  
Vol 216 (3) ◽  
pp. 701-707 ◽  
Author(s):  
J E Seely ◽  
A E Pegg
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Enzyme Protein ◽  
Rapid Loss ◽  
Immunoreactive Protein ◽  
Full Effect

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.

scholarly journals Detection of gene expression and enzyme activity of Cytochrom P450 arom (aromatase) in preantral and early antral bovine follicles depending on culture conditions in vitro

2006 ◽  
Vol 49 (1) ◽  
pp. 29-40
Author(s):  
R. Pöhland ◽  
S. Lenz ◽  
J. Vanselow ◽  
W. Tomek
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Culture Medium ◽  
Culture Conditions ◽  
Enzyme Protein ◽  
Cytochrom P450 ◽  
Realtime Pcr ◽  
Follicle Size ◽  
Estradiol Synthesis

Abstract. This study reveals that cultivation of preantral and early antral (< 500 μm) follicles in culture medium containing FCS results in an expression of cytochrome P450 arom. (aromatase). The enzymatic activity of aromatase, measured in terms of the estradiol synthesis, was proved to be present in follicles greater than 100 μm, could further be stimulated by FSH in follicles greater than 300 μm diameter. The enzyme protein and the gene expression were studied by means of western blot and immunohistochemistry as well as by means of realtime PCR. Neither the protein nor the corresponding mRNA could be found in uncultivated follicles and in FCS-free cultivated follicles. Estradiol synthesis could not be determined under FCS-free conditions. The expression of the FCS effect was dependent on the follicle size.

scholarly journals Phosphopyruvate carboxylase induction by l-tryptophan. Effects on synthesis and degradation of the enzyme

1973 ◽  
Vol 136 (2) ◽  
pp. 259-264 ◽  
Author(s):  
F. J. Ballard ◽  
M. F. Hopgood
Keyword(s):  
Enzyme Activity ◽  
Specific Antibody ◽  
Actinomycin D ◽  
Relative Rate ◽  
Enzyme Synthesis ◽  
Enzyme Protein ◽  
Carboxylase Activity ◽  
Twofold Increase ◽  
Synthesis And Degradation

1. The administration of l-tryptophan to fed rats produces a twofold increase in hepatic phosphopyruvate carboxylase activity that represents a comparable increase in enzyme protein. With specific antibody against the enzyme we have shown that the increase in phosphopyruvate carboxylase is partially mediated via an actinomycin D-sensitive increase in enzyme synthesis. 2. In starved animals tryptophan increases the enzyme activity without any change in the relative rate of phosphopyruvate carboxylase synthesis. In this condition degradation of the enzyme is retarded by tryptophan by a mechanism that is not prevented by cycloheximide.

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

1987 ◽  
Vol 65 (9) ◽  
pp. 776-782 ◽  
Author(s):  
Brenda Allan ◽  
Andrew M. Kropinski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Rna Polymerase ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subunit Structure ◽  
Partial Activity ◽  
Inhibited Enzyme

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having β′ (157 000), β (148 000), σ (87 000), and α2 (45 000) subunits as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+ produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5–9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 °C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.

scholarly journals The effects of salicylate on the activity of rat liver tryptophan pyrrolase in vitro and in vivo

1971 ◽  
Vol 123 (2) ◽  
pp. 171-174 ◽  
Author(s):  
A. A.-B. Badawy ◽  
M. J. H. Smith
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Binding Sites ◽  
Actinomycin D ◽  
Sodium Salicylate ◽  
Progressive Increase ◽  
Tryptophan Pyrrolase ◽  
Enzyme Ratio

1. Salicylate, in concentrations of 0.05mm and above, inhibits the basal activity of tryptophan pyrrolase in homogenates of rat liver and the activity induced by cortisol but not that induced by tryptophan. The inhibition is abolished by adding haematin to the reaction mixtures. 2. The intraperitoneal injection of 400mg of sodium salicylate/kg in the rat causes a decrease in the tryptophan pyrrolase activity in the liver at 30min, the activity is restored to normal at 2h, increases to sixfold after 5h and returns to the basal value at 12h. The activation of the enzyme by salicylate is prevented by the administration of cycloheximide but not by pretreatment with actinomycin D. The effects of the combined injection of salicylate and cortisol are additive, whereas those of salicylate plus tryptophan are not. The injection of salicylate causes a progressive increase in the holo-/apo-enzyme ratio and an increased content of tryptophan in the liver over a period of 3h. 3. It is suggested that salicylate inhibits tryptophan pyrrolase activity in vitro and in vivo by interacting with iron protoporphyrins and causes a later enhancement of the enzyme activity in vivo by a mechanism involving the release of tryptophan from its binding sites on circulating albumin and on other proteins.

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